KR101555872B1 - Composition comprising Diospyros lotus L. leaf extract for anti-allergy or anti-pruritic - Google Patents

Abstract

The present invention relates to a composition containing gourd leaf extract, and more particularly, to a composition containing gourd leaf extract as an effective ingredient and having excellent antiallergic effect. To an anti-itching effect.

Description

A composition for anti-allergic or anti-itching containing gourd leaf extracts (Composition comprising Diospyros lotus leaf extract for anti-allergy or anti-pruritic)

The present invention relates to a composition containing a gomex leaf extract, and more particularly to a composition containing a gomex leaf extract as an active ingredient and exhibiting excellent antiallergic effect and anti-itching effect.

Allergic diseases include allergic skin diseases such as autoimmune disease, collagen disease, etc., including anaphylactic shock, asthma, allergic rhinitis and allergic skin diseases. Globally, 22% are exposed to allergic diseases. The cause of the allergic diseases explosively increases is the increase of allergen caused by environmental pollution, dietary habits and changes in the living environment. Skin diseases that can be sensitized by allergens include contact dermatitis ), Urticaria including eczema such as atopic dermatitis, seborrheic dermatitis, and xerotic dermatitis. In particular, if allergens are sensitized to skin in infants and children, they may be accompanied by itching and inflammation. If they are constantly sensitized to allergens, their symptoms will progress to atopic dermatitis, which is repeatedly exacerbated and alleviated. It can cause disability.

Atopic dermatitis caused by childhood can lead to allergic diseases such as allergic rhinitis, asthma, and conjunctivitis, if it is an atopic or allergic march unless adequate prevention and management is provided. In other words, if allergens are sensitized to the skin, the skin rash and itch may accelerate the inflammatory reaction and cause atopic dermatitis. If the allergen is continuously sensitized to the skin and the respiratory tract, the respiratory diseases such as asthma and allergic rhinitis May develop and may migrate around the eyeball, causing allergic conjunctivitis.

Immunological features of allergic dermatitis include acute and chronic inflammatory responses due to immune disorders such as overactive activation of mast cells including defective development of co-induced cells such as type 2 helper T (Th2) (acute / chronic inflammatory response). Atopic dermatitis is accompanied by inflammation and severe itching accompanied by a collapse of the skin barrier, which makes the symptoms worse.

Mast cells are known to be a key participant in the development of acute and chronic inflammatory diseases in most allergic diseases, including atopic dermatitis. When mast cells are activated, they release inflammatory cytokines such as tumor necrosis factor (TNF) -α, interleukin (IL) -1 and IL-6, as well as pruritogens such as histamine . Thus, inflammatory cytokines derived from mast cells further promote the inflammatory reaction, while itching substances such as histamine further promote itching, thereby causing skin barrier disruption. Thus, identifying substances that can inhibit inflammatory cytokines and itch-mediated substances without side effects will be effective in controlling allergic diseases, including atopic dermatitis.

Goomae ( Diospyros lotus L.) is a deciduous plant that is naturally native to Asia, including Korea and China. In the field of traditional medicines, gourm, a mature fruit, has been used for calming, analgesic, astringent and constipation treatment. Goyom's biochemical components have been reported fatty acids, sugars, flavonoids and non-volatile substances. Recently, there have been reports of anticoagulation, brain cell protection, antioxidant and anticancer effects of blood. However, there is no report on antiallergic and anti-itch against guillae leaf extract.

Accordingly, the inventors of the present invention have found that, when natural substances capable of inhibiting inflammatory cytokines and itch-mediated substances without side effects are studied, the gourd leaf extract significantly inhibits inflammatory cytokines and itching substances without toxicity to cells, .

Accordingly, it is an object of the present invention to provide a composition containing an extract of Gomex leaf as an active ingredient, exhibiting excellent antiallergic effect and anti-itching effect.

In order to attain the above object, the present invention provides a composition comprising an extract of Gomex leaf as an active ingredient.

The present invention also provides a composition for anti-allergy comprising an extract of Goat Leaf as an active ingredient.

The present invention also provides a composition for anti-itching comprising an extract of Goat Leaf as an active ingredient.

Hereinafter, the present invention will be described more specifically.

The goat's leaf extract used in the present invention is a gourd tree ( Diospyros lotus L.). Extraction of the goat's leaf extract can be carried out according to a conventional extraction method in the art. For example, high temperature extraction at 80 DEG C or higher, low temperature extraction at 25 DEG C or lower, ultrasonic extraction, immersion extraction, or extraction with an organic solvent such as benzene or ethanol, preferably at 80 to 100 DEG C for 10 minutes, Extraction can be performed.

The present invention provides a method of inhibiting the activity of mast cells and inhibiting the production of inflammatory cytokines such as TNF-α, IL-1β, IL-4, IL-6 and IgE, It suppresses the release of the same itching substance to control the inflammatory reaction caused by the allergic reaction. It is effective in controlling allergic diseases including atopic dermatitis by suppressing the itching.

The composition according to the present invention may be a pharmaceutical composition. In the pharmaceutical composition according to the present invention, the term " effective ingredient " is intended to directly or indirectly express the efficacy or effect of the drug by inherent pharmacological action (Including pharmacologically active ingredients, etc., of which there are no known pharmacological active ingredients, etc.).

The pharmaceutical compositions according to the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral external preparations, suppositories and sterilized injection solutions, Can be used.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition according to the present invention is prepared by using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used in the case of formulation. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, oral, rectal or intravenous, muscular, subcutaneous, intracerebral or intracerebroventricular injections and directly to the skin. The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the condition and body weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer the goat's leaf extract of the present invention at a dose of 25 mg / kg to 1,000 mg / kg per day, preferably 200 mg / kg to 500 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In the pharmaceutical composition according to the present invention, the guayome leaf extract may be contained in an amount of 0.01 to 10% by weight based on the total weight of the pharmaceutical composition. When the content is less than 0.01% by weight, the effect is insignificant. When the content is more than 10% by weight, the effect is not significantly different from the increase of the content.

The composition according to the present invention is useful for treating or controlling various allergic diseases including atopic dermatitis because it has excellent effect of inhibiting inflammatory cytokines and itch-mediated substances without toxicity or side effects, Lt; / RTI >

FIG. 1 is a graph showing the effect on the mast cell viability of the goat's leaf extract according to the present invention.
FIG. 2 is a graph showing the inhibitory effect on the proinflammatory cytokine (TNF-.alpha., IL-1.beta.) Production of guacole leaf extract according to the present invention.
FIG. 3 is a graph showing inhibitory effect of histone release on gum root leaf extract according to the present invention.
FIG. 4 is a graph showing the effect of inhibiting itching in the extract of goat's worms according to the present invention.
FIG. 5 is a graph showing skin lesion (A), skin texture thickness (B), leukocyte infiltration (C) and mast cell infiltration (D) results in DNFB inflammation model mice.
FIG. 6 is a graph showing the amounts of IgE and IL-4 production in DNFB inflammation model mice. FIG.
FIG. 7A is a photograph showing the skin condition of a normal control group and an extract-administered group mouse 6 hours after the last DNFB attack, and B is an X100 magnification photograph of a mouse skin tissue stained with H & E showing the leukocyte infiltration state and C Is an X400 magnification image stained with toluidine blue and shows the infiltration state of mast cells.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited by these examples or test examples.

[Referential Example 1] Preparation of Goat Leaf Extract

D. lotus L. was collected on June 30, 2012 at Shingi Village, Suwang-ri, Gugong-myeon, Jinan-gun, Jeollabuk-do. The identification of Goyom leaf was confirmed by Professor Kim Hong-joon of Woosuk University School of Oriental Medicine, Hwangseok University. The Goyom leaf sample (# 2012-0630) is kept in the Health Management Laboratory of the College of Alternative Medicine at Jeonju University. The collected gom- yle leaves were immediately washed with distilled water, steamed for 5 minutes, dried at room temperature using a fan, and finally dried at 40 ° C for 12 hours in a dryer. The extract was filtered through a 0.45 μm filter and dried in a freeze dryer (Model, EYELA FDU-2100, Japan) to recover 19.5 g of the extract. And stored at -20 ° C.

[Reference Example 2] Experimental animals and reagents

Seven-week-old hairless mice and ICR mice and 10-week-old Sprague Dawley rats raised in an aseptic environment were purchased from Central Experimental Animal Co. (Seoul, Korea) and fed with feed and water for one week And then used for experiments. The incubation environment consisted of day and night cycles for 12 hours, and the temperature (20-22 ℃) and humidity (50-60%) were kept constant and tested according to the provisions of the Laboratory Animal Committee.

An ELISA kit including Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1), il-6 and histamine was purchased from R & D Systems (Minneapolis, USA). Compound 48/80, histamine, methysergide maleate (PMA), calcium ionophore A23187, 2,4-dinitrofluorobenzene (DNFB), dimethyl sulfoxide (DMSO), penicillin / streptomycin, hematoxylin and eosin, toluidine blue and other products such as percoll, compound 48/80, phorbol 12-myristate 13- The reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[Reference Example 3] Isolation of peritoneal mast cells (RPMCs)

Rat peritoneal mast cells (RPMCs) were isolated from male Sprague Dawley rats prepared in Reference Example 2 by the method of Martynova et al. More specifically, it was anesthetized the rats with ether (ether), and then calcium-free HEPES-Tyrode buffer ( 10 mM Hepes, 113 mM NaCl, 4.7 mM KCl, 2.13 mM MgCl 2, 0.6 mM NaH 2 PO 4, 10 mM glucose, pH 7.4) was injected into the abdominal cavity and the abdominal cavity was gently massaged for about 90 seconds. The abdominal cavity was carefully opened, and the cell suspension was taken using a Pasteur pipette. The peritoneal mast cells (RPMCs ). In the experiment, peritoneal mast cells obtained from 5 to 10 rats were mixed and used.

[Test Example 1] Effect on survival rate of mast cell

The cell survival rate was measured by the MTT assay in order to determine the effect of the goat's leaf extract on mast cell viability.

More specifically, 5 × 10 ⁵ / ml of rat peritoneal mast cells (RPMCs) isolated in Reference Example 3 was inoculated into a 24-well culture vessel, and the goat's leaf extract according to Reference Example 1 / Ml, 50 占 퐂 / ml, 100 占 퐂 / ml and 200 占 퐂 / ml. Two hours after the treatment of goat's leaf extract, PMA 30 ng / ml and A23187 10 μM, known as RPMCs degassed inducers, were mixed and treated with MTT solution 5 mg / ml for 12 hours. , The supernatant was removed, the formazan product was dissolved in dimethyl sulfoxide (DMSO), transferred to a 96-well plate, and absorbance was measured at 540 nm using an ELISA reader (Molecular Divices, USA) When the absorbance was 100, the absorbance of the experimental group was expressed as a percentage and the cell viability was calculated. The results are shown in FIG.

FIG. 1 (A) shows cell survival rates according to the MTT assay method in the same manner as above except that PMA and A23187 administration were performed 24 hours after the treatment of extracts treated with gonium leaf extract alone at peritoneal mast cells As a result, it can be confirmed that the gomule leaf extract according to the present invention is free from cytotoxicity.

In the result of FIG. 1B, the cell viability was about 83% when treated with PMA and A23187, which are known as degranulation inducers of mast cells, and the cell survival rate was lowered compared with the normal control group (untreated group) The cell viability is increased in a concentration-dependent manner. Especially, the cell survival rate was significantly increased at the concentration of 100 ㎍ / ㎖ or more of goat leaf extract (p <0.05, p <0.01).

[Test Example 2] Inhibitory effect on proinflammatory cytokine production

The amount of proinflammatory cytokines TNF-α and IL-1β was measured by ELISA in order to confirm the inhibitory effect of guayome leaf extract on the proinflammatory cytokine production according to the present invention. More specifically, the goat's leaf extract according to Reference Example 1 was added to 5 x 10 &lt; ⁵ &gt; / ml of peritoneal mast cells (RPMCs) isolated in Reference Example 3 in an amount of 0 g / ml, 50 g / After 2 hours of treatment at a concentration of 200 μg / ml, 30 ng / ml of PMA and 10 μM of A23187 were treated and after 12 hours, the cell supernatant was obtained. The cell supernatants were each injected into a plate coated with anti-mouse TNF-α and anti-mouse IL-1β antibodies, washed, and then washed with a secondary antibody (horseradish peroxidase- Rabbit Anti-mouse IgG) was injected and reacted, and tetramethylbenzidine (TMB) was injected as a chromogenic substrate, and the reaction was measured by an ELISA reader (Molecular Divices, USA). The standard curve for each concentration of each substance was determined and the amount of the substance contained in the cell supernatant was calculated. The results are shown in FIG.

In the results of FIG. 2, the production of TNF-α and IL-1β was significantly increased (p <0.001) in comparison with the treatment with PMA and A23187 as compared with the control group without any drug (untreated group) The production of TNF-α and IL-1β was inhibited in a concentration-dependent manner in the experimental group treated with goat's leaf extract. TNF-α showed a significant inhibitory effect (p <0.05 and p <0.01) in the treatment group of 100 μg / ㎖ or more. IL-1β was significantly inhibited at a concentration of 50 μg / ml or more (p <0.05, p <0.01 and p <0.001).

[Test Example 3] Histamine release inhibitory effect

In order to confirm the release inhibitory effect on histamine, which is the core substance of itching, in the gomoma leaf extract according to the present invention, the following experiment was conducted.

50 μg / ml, 100 μg / ml and 200 μg / ml, respectively, of the goat's leaf extract according to Reference Example 1 were added to 5 × 10 ⁵ / ml of peritoneal mast cells (RPMCs) After 10 minutes at 37 ° C, compound 48/80 was treated with 0.5 μg / ml and allowed to stand for 30 minutes. To stop the reaction, the culture tube was placed in ice water and sufficiently cooled, centrifuged at 1,200 rpm for 15 minutes The supernatant was separated and the amount of histamine was measured. The amount of histamine was measured by injecting the supernatant into a plate coated with anti-histamine antibody and reacting with a rabbit anti-histamine antibody, which had acetylcholinesterase attached thereto as a secondary antibody, -mouse IgG was injected and reacted. Then, 5,5'-dithiobis- (2-nitrobenzoic acid) (DTNB) was reacted as a chromogenic substrate to generate color And the amount of histamine was measured and quantified by an ELISA reader (Molecular Divices, USA) according to the ELISA method provided by R & D. The results are shown in FIG.

3, when the RPMCs were stimulated with compound 48/80, the amount of histamine released was significantly increased (p <0.001) as compared with the control group (p <0.001), and when the goat's leaf extract according to the present invention was treated, (P <0.001). In particular, the effect of inhibiting histamine release was remarkable in the group treated with 200 μg / ml (p <0.001).

[Test Example 4] Inhibitory effect on itching

The following experiments were conducted to confirm the inhibitory effect of goat's leaf extract on itching according to the present invention.

Five ICR mice prepared in Reference Example 2 were put into each of the transparent acrylic cages (20 x 26 x 13 cm) one by one for each test group and allowed to stand in the same environment for 30 minutes to stabilize them. Then, physiological saline was orally administered as a control group, and Oral administration of goat's leaf extract according to Reference Example 1 was orally administered at 0 mg / kg, 50 mg / kg, 100 mg / kg and 200 mg / After injection of compound 48/80 at a concentration of 50 / / site or 100 / / site of histamine at a height of between both shoulders such as a mouse, sc injection was induced.

The mice injected with the itch inducer were immediately photographed for 60 minutes using a micro-camera (ONCCTV, Seoul, Korea) according to the method of Mihara, and the number of scratching of the area where the itch inducer was implanted was determined by double blind The results are shown in FIG. Experiments on each inducer were conducted on different days and the mice used in each experiment were used once.

In the results of FIG. 4, compound 48/80 or histamine-subcutaneously injected mice significantly increased the number of scratches compared to the normal control group (p <0.001). However, it was confirmed that the experimental group treated with goat's leaf extract had a concentration-dependent inhibition of itching (p <0.05, p <0.01 and p <0.001).

[Test Example 5] Anti-inflammatory effect of DNFB inflammation model mouse

The following experiment was conducted to confirm whether the gourd leaf extract according to the present invention has an inhibitory effect on skin lesion inhibition and related mediator production in a DNFB-induced allergic model mouse as a chemical antigen.

The dorsal skin of a 7 week old male nude mouse prepared in Reference Example 2 was treated with 0.15% 2,4-dinitrofluorobenzene (DNFB) using a solvent prepared from 3: 1 acetone: olive oil 100 μl each was applied to the skin on days 1 and 4 and sensitized. From the second sensitization day 4, the control group was injected with 0.2 ml / mouse of physiological saline solution into the abdominal cavity once a day until the end of the experiment. As the experimental group, the goat's leaf extract according to Reference Example 1 was administered at 50 mg / kg, 100 mg / ㎎ / ㎏ in the abdominal cavity once a day until the end of the experiment. On the 7th, 10th and 13th days after the first sensitization, the mice were challenged with the same dose at the sites sensitized with 0.2% DNFB at intervals of 3 days.

<Skin lesion evaluation>

The skin condition was examined by taking pictures for 4 weeks at weekly intervals from week 0. After 6 hours after the final attack, skin dryness, scaling, erosion, excoriation, and bleeding were checked to see if there was no lesion at 0 point, (Mild), moderate (middle), and severe (severe). The total score was assigned to each step. The results are shown in FIG. 5A.

&Lt; Measurement of skin tissue thickness &

5 hours after the final attack, the thickness of the dorsal skin tissue was measured using a digital caliper (Mitutoyo, Japan) and is shown in Fig. 5B.

&Lt; Evaluation of inflammatory cells &

The mice were sacrificed and about 5 x 5 mm of skin tissue was extracted and fixed with 4% paraformaldehyde (pH 7.4), paraffin blocks were prepared, and the skin tissues were cut into longitudinal sections at intervals of 5 쨉 m. (H & E) and 400X (H & E) were observed while histologically examined and leukocyte and mast cell infiltration were observed with hematoxylin & eosin (H & E) or toluidine blue and observed at low magnification (40X) (Figure 7B) and mast cells (Figure 7C) by taking pictures (toluene blue) in the microscope field of view (B and C, respectively, in Figures 7).

The number of infiltrating leukocytes was counted in a 400X microscope field (Olympus, Japan), and the results are shown in Fig. 5C. The number of mast cells was counted and the result is shown in Fig.

&Lt; Measurement of IgE and IL-4 production amount >

Five hours after the final attack, blood was collected from the portal vein of the mouse, and serum was taken. The serum was injected into plates coated with anti-IgE and anti-IL-4 antibodies, (Rabbit anti-mouse IgG), which is a secondary antibody specifically acting on the antibody-4 antibody, was injected and reacted. Then, tetramethylbenzidine ; TMB) were injected and reacted and measured with an ELISA reader (Molecular Divices, USA). The amount of IgE and IL-4 production was measured and quantified by ELISA, and the results are shown in FIG.

In the results of FIGS. 5 to 7, the normal control group had no change in the skin tissue after the experiment, but in the control group sensitized with DNFB, severe skin lesions and inflammation were accompanied (Fig. 7A, Fig. 5A) acanthosis, hyperkeratosis, wound and hemorrhage were very severe (Fig. 7) and their thickness was significantly increased (Fig. 5B). However, in the experimental group treated with DNFB and guayome leaf extract, the symptom was alleviated compared with the control group sensitized with DNFB, and the symptom was remarkably improved in the experimental group treated with 200 mg / kg Goat's leaf extract ( p &lt; 0.001).

In order to examine leukocyte and mast cell infiltration effects, leucocyte infiltration (Fig. 7B) and obesity were measured by DNFB-sensitized mice in H & E and toluidine blue. Leukocyte infiltration and mast cell activity were inhibited in the experimental group treated with goat yam leaf extract. In particular, the experimental group treated with 200 mg / kg goat yam leaf extract showed leukocyte infiltration and mast cell activity (Fig. 7C) (Fig. 7B, 7C and Fig. 5C, 5D).

In addition, in the results of FIG. 6, IL-4 and IgE production was significantly increased in the control group sensitized with DNFB only, whereas the production of IL-4 and IgE was significantly inhibited in the experimental group treated with goatee leaf extract, Especially, the experimental group treated with 200 ㎎ / ㎏ goat's leaf extract showed significant improvement (p <0.001).

Claims (5)

A composition for anti-allergy characterized by containing Gouomer ( Diospyros lotus L.) leaf extract as an active ingredient and inhibiting inflammatory cytokine production or inhibiting histamine release. A composition for anti-itching characterized by containing Goumarz ( Diospyros lotus L.) leaf extract as an active ingredient and inhibiting the production of inflammatory cytokines or inhibiting histamine release. delete The composition according to claim 1 or 2, wherein the composition is for external application for skin, for oral administration or for injection. The composition according to claim 1 or 2, wherein the gomace leaf extract comprises 0.01 to 10% by weight based on the total weight of the composition.

Images